Tumor growth and progression requires development of a new vascular supply. Many current anti-angiogenic therapies target VEGF, but targeting VEGF alone is not universally effective in combating angiogenesis induced by tumors in different organs or at various stages of the disease. Thus more global approaches to inhibit angiogenesis are needed. My laboratory has defined roles for the homeobox (Hox) master transcription factors, HoxD3, HoxA3 and HoxB3 in coordinately regulating endothelial cell (EC) invasion, proliferation, and adhesion during angiogenesis. In addition we have also demonstrated that other Hox factors, namely HoxD10 and HoxA5 act in a dominant manner to maintain cultured EC in a quiescent, differentiated state and resist angiogenic stimuli. To explore the anti-angiogenic and anti-tumorigenic potential of Hox genes in vivo, we will focus on HoxA5, which is highly expressed in quiescent differentiated EC, but expression is reduced in activated tumor endothelium. Ectopic expression of HoxA5 in cultured EC impairs migration, down-regulates several pro-angiogenic genes including VEGFR2, ephrin A1 and IL-6 and significantly increases levels of anti-angiogenic factors including Thrombospondin-2 (TSP-2). HoxA5 also stabilizes EC adherens junctions and reduces endothelial cell permeability. Our preliminary evidence suggests that EC HoxA5 may also suppress recruitment/infiltration of pro-angiogenic bone marrow derived cells (BMDC). Thus, we hypothesize that maintaining high levels of HoxA5 selectively in EC in the tumor microenvironment in vivo will stabilize the vasculature, inhibit angiogenesis and BMDC recruitment and impair tumor progression. We have developed a transgenic mouse model in which expression of HoxA5 is largely restricted to EC through TIE-2 driven expression of a tetracycline activator protein and HoxA5 can be activated in angiogenic conditions by removal of tetracycline. These mice have been crossed with 2 established de novo models of skin (K14-HPV16) and mammary MMTV-PyMT) carcinogenesis for further analysis of EC HoxA5 in reducing tumor angiogenesis and progression in complex and dynamic tumor microenvironments. We will also evaluate how EC HoxA5 influences recruitment of pro-angiogenic BMDC in these models. Finally we will develop methods to therapeutically restore HoxA5 expression in tumor tissues in vivo and explore the ability of HoxA5 to potentially coordinate the behavior of endothelial, epithelial cells and recruitment of BMDC within tissues to identify new approaches to treat tumors and their reactive stroma. PUBLIC HEALTH RELEVANCE: These studies will investigate the whether Hox genes which maintain a quiescent, mature phenotype in vascular endothelial cells, can be sustained in angiogenic environments and thus block tumor angiogenesis and progression. We will focus on the vascular stabilizing transcription factor, HoxA5, and using a novel transgenic mouse model, will sustain its expression in tumor endothelial cells in vivo to determine whether this impairs angiogenesis and ultimately limits tumor progression. Moreover, as this gene is normally expressed in quiescent resting vessels, but absent in tumor vessels, sustained expression would not interfere with normal vascular function. In addition we will develop two novel approaches to apply HoxA5 therapeutically to both skin and mammary tumors to further demonstrate its clinical applicability. Finally we will explore the notion that HoxA5 may act to coordinately stabilize the vasculature as well as the tumor epithelium and the pro-tumorigenic immune response thus represent a means to target the entire tumor microenvironment.